Project 3 Abstract Expanded genotyping and next generation sequencing in ever-larger disease cohorts has been remarkably successful in identifying novel loci, genes and alleles that drive pathology and/or confer susceptibility. However, these efforts have encountered three major obstacles. First, association studies with some notable examples have been hampered by the difficulty in transitioning efficiently from mapping loci to identifying contributory genes and alleles. Second, ultra-rare or private Mendelian mutations have been challenging to identify in sufficient numbers to link them robustly to phenotype. Third, an overwhelming majority of variation identified in the genetically heterogeneous gonadotropin-releasing hormone (GnRH) disorders are non- synonymous changes for which allele pathogenicity is difficult to infer using genetic arguments alone. One reason for these impediments is the presence of the large amount of rare variation that exists in human populations and the current bioinformatic tools remain limited in predicting with accuracy which fraction of this variation is functional and phenotypically relevant, Together with our colleagues in Projects 1 and 2, we have intersected genetic, genomic and functional tools to overcome some of these challenges and have begun to inform the genetic architecture of disorders of reproductive development. Our past synergistic efforts led to the identification of RNF216 and OTUD4 as the first genes mutated in isolated hypogonadotropic hypogonadism (IGD) in individuals with Gordon-Holmes syndrome establishing a ?module? of dysfunction, namely the ubiquitin-proteasome pathway in this disorder. Complementary to this work, systematic in vivo functional assessment of an allelic series in CHD7 allowed us to isolate the non-Mendelian contribution of this locus to reproductive disorders of the GnRH axis under a mutational burden hypothesis that was otherwise refractory to classical statistical tools. In the next phase of these investigations, Project 3 will expand its role as a bridge between the ongoing and successful Mendelian gene discovery efforts (Project 1) and the state-of-the-art complex trait association approaches (Project 2). Project 3 will assess the pathogenicity of novel candidate genes and loci from Projects 2 and 3 respectively. It will also annotate non-synonymous coding variants discovered as part of this process in IGD patients and large populations as a means of determining the mutational burden in affected individuals. We will also utilize the in vivo models we generate to model oligogenic phenomena observed in patient cohorts. In parallel, Project 3 will isolate pure cellular populations relevant to GnRH biology from zebrafish models to generate transcriptional networks that will inform the studies of the overall Center.